Compositions and methods for reverse transcriptase-polymerase chain reaction (RT-PCR)

ABSTRACT

The present invention is directed to compositions and methods useful for the amplification of nucleic acid molecules by reverse transcriptase-polymerase chain reaction (RT-PCR). Specifically, the invention provides compositions and methods for the amplification of nucleic acid molecules in a simplified one- or two-step RT-PCR procedure using combinations of reverse transcriptase and thermostable DNA polymerase enzymes in conjunction with sulfur-containing molecules or acetate-containing molecules (or combinations of such sulfur-containing molecules and acetate-containing molecules), and optionally bovine serum albumin. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules. The invention also is useful in the rapid production and amplification of cDNAs which may be used for a variety of industrial, medical and forensic purposes.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/042,629, filed Apr. 3, 1997, the disclosure of whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is in the fields of molecular and cellularbiology. The invention is particularly directed to compositions andmethods useful for the amplification of nucleic acid molecules byreverse transcriptase-polymerase chain reaction (RT-PCR). Specifically,the invention provides compositions and methods for the amplification ofnucleic acid molecules in a simplified one- or two-step RT-PCR procedureusing-combinations of reverse transcriptase and thermostable DNApolymerase enzymes in conjunction with sulfur-containing molecules oracetate-containing molecules (or combinations of sulfur-containingmolecules and acetate-containing molecules) and optionally bovine serumalbumin. The invention thus facilitates the rapid and efficientamplification of nucleic acid molecules and the detection andquantitation of RNA molecules. The invention also is useful in the rapidproduction and amplification of cDNAs (single-stranded anddouble-stranded) which may be used for a variety of industrial, medicaland forensic purposes.

BACKGROUND OF THE INVENTION

[0003] Reverse Transcription of RNA

[0004] The term “reverse transcriptase” describes a class of polymerasescharacterized as RNA-dependent DNA polymerases. All known reversetranscriptases require a primer to synthesize a DNA transcript from anRNA template. Historically, reverse transcriptase has been usedprimarily to transcribe mRNA into cDNA which can then be cloned into avector for further manipulation.

[0005] Avian myoblastosis virus (AMV) reverse transcriptase was thefirst widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys.Acta 473:1 (1977)). The enzyme has 5′-3′ RNA-directed DNA polymeraseactivity, 5′-3′ DNA-directed DNA polymerase activity, and RNase Hactivity. RNase H is a processive 5′ and 3′ ribonuclease specific forthe RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide toMolecular Cloning, New York: Wiley & Sons (1984)). Errors intranscription cannot be corrected by reverse transcriptase because knownviral reverse transcriptases lack the 3′-5′ exonuclease activitynecessary for proofreading (Saunders and Saunders, Microbial GeneticsApplied to Biotechnology, London: Croom Helm (1987)). A detailed studyof the activity of AMV reverse transcriptase and its associated RNase Hactivity has been presented by Berger et al., Biochemistry 22:2365-2372(1983).

[0006] Another reverse transcriptase which is used extensively inmolecular biology is reverse transcriptase originating from Moloneymurine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271-279(1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985). M-MLVreverse transcriptase substantially lacking in RNase H activity has alsobeen described. See, e.g., U.S. Pat. No. 5,244,797.

[0007] PCR Amplification of RNA

[0008] Reverse transcriptases have been extensively used in reversetranscribing RNA prior to PCR amplification. This method, often referredto as RNA-PCR or RT-PCR, is widely used for detection and quantitationof RNA

[0009] To attempt to address the technical problems often associatedwith RT-PCR, a number of protocols have been developed taking intoaccount the three basic steps of the procedure: (a) the denaturation ofRNA and the hybridization of reverse primer; (b) the synthesis of cDNA;and (c) PCR amplification. In the so-called “uncoupled” RT-PCR procedure(e.g., two-step RT-PCR), reverse transcription is performed as anindependent step using the optimal buffer condition for reversetranscriptase activity. Following cDNA synthesis, the reaction isdiluted to decrease MgCl₂ and deoxyribonucleoside triphosphate (dNTP)concentrations to conditions optimal for Taq DNA Polymerase activity,and PCR is carried out according to standard conditions (see U.S. Pat.Nos. 4,683,195 and 4,683,202). By contrast, “coupled” RT-PCR methods usea common or compromised buffer for reverse transcriptase and Taq DNAPolymerase activities. In one version, the annealing of reverse primeris a separate step preceding the addition of enzymes, which are thenadded to the single reaction vessel. In another version, the reversetranscriptase activity is a component of the thermostable Tth DNApolymerase. Annealing and cDNA synthesis are performed in the presenceof Mn⁺⁺ then PCR is carried out in the presence of Mg⁺⁺ after theremoval of Mn⁺⁺ by a chelating agent. Finally, the “continuous” method(e.g., one-step RT-PCR) integrates the three RT-PCR steps into a singlecontinuous reaction that avoids the opening of the reaction tube forcomponent or enzyme addition. Continuous RT-PCR has been described as asingle enzyme system using the reverse transcriptase activity ofthermostable Taq DNA Polymerase and Tth polymerase and as a two-enzymesystem using AMV-RT and Taq DNA Polymerase wherein the initial 65° C.RNA denaturation step was omitted.

[0010] Attempts to streamline the process of RT-PCR have not been easy,and several reports have documented an interference between reversetranscriptase and thermostable DNA polymerase Taq when used incombination in a single tube RT-PCR resulting in low sensitivity or lackof results. For example, there has been at least one report of a generalinhibition of Taq DNA polymerase when mixed with reverse transcriptasesin one-step/one tube RT-PCR mixtures (Sellner, L. N., et al., Nucl.Acids Res. 20(7):1487-1490 (1992)). This same report indicated that theinhibition was not limited to one type of RT: both AMV-RT and M-MLV-RTinhibited Taq DNA polymerase and limited the sensitivity of RT-PCR.Under the reaction conditions used in the Sellner et al. studies (67 mMTris-HCl, pH 8.8, 17 mM (NH₄)₂ SO₄, 1 mM β-mercaptoethanol, 6 μM EDTA,0.2 mg/ml gelatin), the degree of Taq polymerase inhibition was found toincrease with increasing RT concentration, up to a ratio ofapproximately 3 units of RT:2 units of Taq DNA polymerase beyond whichTaq polymerase was rendered completely inactive.

[0011] Other reports describe attempts to develop conditions forone-step RT-PCR reactions. For example, the use of AMV-RT for one-stepRT-PCR in a buffer comprising 10 mM Tris-HCl, (pH 8.3), 50 mM KCl, 1.5mM MgCl₂, and 0.01% gelatin has been reported (Aatsinki, J. T., et al,BioTechniques 16(2):282-288 (1994)), while another report demonstratedone-step RT-PCR using a composition comprising AMV-RT and Taq DNApolymerase in a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KCl,0.01% gelatin and 1.5 mM MgCl₂ (Mallet, F., et al., BioTechniques18(4):678-687 (1995)). Under the reaction conditions used in the latterreport, substitution of M-MLV-RT (RNase H⁺ or RNase H⁻ forms) for AMV-RTshowed the same activity in the continuous RT-PCR reaction.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention is generally directed to compositions andmethods useful for one-step/one-tube RT-PCR, preferably using M-MLV-RT,or its RNase H-deficient (“RNase H⁻”) derivatives, in combination withone or more DNA polymerases, preferably in the presence ofsulfur-containing molecules or acetate-containing molecules (orcombinations of sulfur-containing molecules and acetate-containingmolecules) to relieve the inhibition of PCR often observed when usingcompositions comprising two or more enzymes having reverse transcriptaseactivity.

[0013] In particular, the invention is directed to methods foramplifying a nucleic acid molecule comprising (a) mixing an RNA templatewith a composition comprising a Moloney murine leukemia virus (M-MLV)reverse transcriptase, which is preferably substantially reduced inRNase H activity and which is most preferably SuperScript I orSuperScript II, in combination with one or more DNA polymerases and oneor more sulfur-containing molecules, such as one or moresulfur-containing buffers, wherein the concentration of sulfur is atleast 18 mM, to form a mixture; and (b) incubating the mixture underconditions sufficient to amplify a DNA molecule complementary to all ora portion of the RNA template. In a related aspect, the invention isdirected to such methods wherein one or more acetate-containingmolecules, such as one or more acetate-containing buffers, issubstituted for or combined with the one or more sulfur-containingmolecules or buffers in step (a) of the above-described methods, whereinthe concentration of the one or more acetate-containing molecules isabout 1 mM to about 500 mM. In preferred such methods, the DNApolymerases used are thermostable DNA polymerases, and most preferablyTne, Tma, Taq, Pfu, Tth, VENT, DEEPVENT, Pwo, Tfl, or a mutant, variantor derivative thereof; most preferred in this aspect of the invention isTaq DNA polymerase.

[0014] In other preferred aspects of the invention the DNA polymerasesmay comprise a first DNA polymerase having 3′ exonuclease activity, mostpreferably a DNA polymerase selected from the group consisting of Pfu,Pwo, DEEPVENT, VENT, Tne, Tma, Kod, and mutants, variants andderivatives thereof, and a second DNA polymerase having substantiallyreduced 3′ exonuclease activity, most preferably a DNA polymeraseselected from the group consisting of Taq, Tfl, Tth, and mutants,variants and derivatives thereof. In additional preferred aspects of theinvention, the unit ratio of the reverse transcriptase to the DNApolymerases is from about 0.2:2 to about 500:2, and in particularlypreferred such aspects the ratio is from about 0.5:2 to about 250:2 orgreater than about 3:2.

[0015] In other preferred aspects of the invention, the concentration ofthe one or more sulfur-containing molecules is at least 18 mM and morepreferably about 20 mM to about 50 mM. The invention is also directed tosuch methods wherein the source of the sulfur-containing molecules is abuffer or a sulfur-containing salt which may be ammonium sulfate,magnesium sulfate, TRIS-sulfate, or manganese sulfate, as well as othersulfur-containing buffers and salts that will be familiar to one ofordinary skill.

[0016] In other preferred aspects of the invention, the concentration ofthe one or more acetate-containing molecules is about 1 mM to about 500mM, and more preferably about 5 mM to about 250 mM, about 10 mM to about200 mM, about 25 mM to about 150mM about 50 mM to about 100 mM, or about60 mM. The invention is also directed to such methods wherein the sourceof the acetate-containing molecules is a buffer or an acetate-containingsalt which may be ammonium acetate, magnesium acetate, TRIS-acetate, ormanganese acetate, as well as other acetate-containing buffers and saltsthat will be familiar to one of ordinary skill.

[0017] The invention is also directed to such methods wherein themixture further comprises one or more nucleotides, preferablydeoxyribonucleoside triphosphates (most preferably dATP, dUTP, dTTP,dGTP or dCTP), dideoxyribonucleoside triphosphates (most preferablyddATP, ddUTP, ddGTP, ddTTP or ddCTP) or derivatives thereof. Suchnucleotides may optionally be detectably labeled (e.g. with aradioactive or nonradioactive detectable label).

[0018] The invention is also directed to such methods wherein themixture further comprises one or more oligonucleotide primers, which arepreferably an oligo(dT) primers, random primers, arbitrary primers ortarget-specific primers, and which is more preferably a gene-specificprimer.

[0019] The invention is also directed to such methods wherein theincubating step comprises (a) incubating the mixture at a temperature(most preferably a temperature from about 35° C. to about 60° C.) andfor a time sufficient to make a DNA molecule complementary to all or aportion of the RNA template; and (b) incubating the DNA moleculecomplementary to the RNA template at a temperature and for a timesufficient to amplify the DNA molecule, preferably via thermocycling,more preferably thermocycling comprising alternating heating and coolingof the mixture sufficient to amplify said DNA molecule, and mostpreferably thermocycling comprising alternating from a first temperaturerange of from about 90° C. to about 100° C., to a second temperaturerange of from about 40° C. to about 75° C., preferably from about 65° C.to about 75° C. In particularly preferred aspects of the invention, thethermocycling is performed greater than 10 times, more preferablygreater than 20 times.

[0020] The invention is also directed to such methods wherein theamplification is not substantially inhibited.

[0021] The invention is also directed to methods for amplifying anucleic acid molecule comprising (a) mixing an RNA template with acomposition comprising a Moloney murine leukemia virus (M-MLV) reversetranscriptase, which is preferably substantially reduced in RNase Hactivity, in combination with one or more DNA polymerases (mostpreferably selected from the group consisting of Tne, Tma, Taq, Pfu,Tth, VENT, DEEPVENT, Pwo, Tfl, and mutants, variants and derivativesthereof), one or more sulfur-containing molecules and one or morepotassium-containing molecules, to form a mixture; and (b) incubatingthe mixture under conditions sufficient to amplify a DNA moleculecomplementary to all or a portion of the RNA template. In a relatedaspect, the invention is directed to such methods wherein one or moreacetate-containing molecules, such as one or more acetate-containingbuffers, is substituted for or combined with the one or moresulfur-containing molecules or buffers in step (a) of theabove-described methods.

[0022] The invention is also directed to methods for amplifying anucleic acid molecule comprising (a) mixing an RNA template with acomposition comprising a Moloney murine leukemic virus (M-MLV) reversetranscriptase and one or more DNA polymerases, wherein the unit ratio ofthe reverse transcriptase to the DNA polymerases is greater then 3:2, toform a mixture; and (b) incubating the mixture under conditionssufficient to amplify a DNA molecule complementary to all or a portionof the RNA template.

[0023] The invention is also directed to compositions comprising aMoloney Murine Leukemic virus (M-MLV) reverse transcriptase, one or moreDNA polymerases and one or more sulfur-containing molecules (wherein thesulfur concentration is at least 18 mM) or one or moreacetate-containing molecules (wherein the acetate concentration is about1 mM to about 500 mM), or combinations of one or more sulfur-containingmolecules and one or more acetate-containing molecules at the aboveconcentrations.

[0024] The invention is also directed to compositions comprising aMoloney Murine Leukemic virus (M-MLV) reverse transcriptase, one or moreDNA polymerases, one or more potassium-containing molecules and one ormore sulfur-containing molecules (wherein the sulfur concentration is atleast 18 mM) or one or more acetate-containing molecules (wherein theacetate concentration is about 1 mM to about 500 mM), or combinations ofone or more sulfur-containing molecules and one or moreacetate-containing molecules at the above concentrations.

[0025] The invention is also directed to compositions comprising aMoloney Murine leukemic virus (M-MLV) reverse transcriptase and one ormore DNA polymerases, wherein the unit ratio of the reversetranscriptase to the DNA polymerases is greater than 3:2.

[0026] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of the following drawings anddescription of the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a photograph of an ethidium bromide (EtdBr)-stained geldemonstrating the inhibition of RT-PCR by reverse transcriptase. Lanes1, 10, 11 and 20 contain 100 bp DNA sizing ladders.

[0028]FIG. 2A is photograph of an EtdBr-stained gel demonstrating theprotective role of bovine serum albumin (BSA) in RT-PCR of β-actin mRNAfrom 100 pg of HeLa total mRNA template. Lane 1 contains a DNA sizingladder, and the arrow indicates the 1026 bp β-actin target sequence.

[0029]FIG. 2B is a photograph of an EtdBr-stained gel demonstrating theprotective role of BSA in RT-PCR of CAT mRNA from 10⁵ copies of CAT mRNAtemplate. Lane 1 contains a DNA sizing ladder, and the arrow indicatesthe 653 bp CAT target sequence.

[0030]FIG. 3 is a photograph of an EtdBr-stained gel demonstrating theperformance of various RTs in sulfate ion- or BSA-containing buffers.Lane 1: 100 bp DNA sizing ladder; Lanes 2-5: 5 units/reaction of AMV-RT;Lanes 6-9: 5 units/reaction of M-MLV-RT; Lanes 10-13: 5 units/reactionof M-MLV-RT (RNase H⁻). Arrow indicates 500 bp CAT target sequence.

[0031]FIG. 4 is a photograph of an EtdBr-stained gel demonstrating theeffect of reverse transcription reaction temperature on RT-PCR productformation. Duplicate samples of 100 ng (lanes 1-6) or 10 ng (lanes 7-12)of total HeLa RNA were reverse transcribed at the indicatedtemperatures, and a 606 bp DNA polymerase III ε subunit target sequence(lanes 1-6) or a 253 bp β-actin target sequence (lanes 7-12) wereamplified by PCR. M: DNA sizing markers.

[0032]FIG. 5 is a photograph of an EtdBr-stained gel demonstratingimproved yield of RT-PCR products obtained by conducting the RT reactionat higher temperatures. Duplicate samples of 10 ng of tobacco total RNA(lanes 1-6) or of 100 ng of total HeLa RNA (lanes 7-12) were reversetranscribed at 45° C. (lanes 1, 2, 7 and 8), 50° C. (lanes 3, 4, 9 and10) or 55° C. (lanes 5, 6, 11 and 12), and a 500 bp GADPH targetsequence (arrowhead; lanes 1-6) or a 1475 bp DNA polymerase III εsubunit target sequence (arrow; lanes 7-12) were amplified by PCR. M:DNA sizing markers; L: 100 bp nucleic acid sizing ladder.

[0033]FIG. 6 is a photograph of an EtdBr-stained gel demonstrating thesensitivity and efficiency of the invention in RT-PCR of large (2-3 kb)products. A 2.78 kb tuberous sclerosis II fragment was amplified fromvarying amounts of total HeLa RNA that was reverse transcribed using thecompositions of the invention without (lanes 1-6) or with (lanes 7-12)the addition of 1 μl of ELONGASE Enzyme Mix, or using a RT kit fromSupplier A (lanes 13-18). Lanes 1, 2, 7, 8, 13 and 14: 1 ng of HeLa RNA;Lanes 3, 4, 9, 10, 15 and 16: 10 ng of HeLa RNA; Lanes 5, 6, 11, 12, 17and 18: 100 ng of HeLa RNA; M: DNA sizing markers.

[0034]FIG. 7 is a photograph of an EtdBr-stained gel demonstrating thesensitivity and efficiency of the invention in RT-PCR of long (>3 kb)products. 100 ng of total HeLa RNA was reverse transcribed using thecompositions of the invention with the addition of 1 μl of ELONGASEEnzyme Mix, and fragments of the adenomatous polyposis coli gene thatwere 7.3 kb (lanes 1, 2), 7.7 kb (lanes 3, 4) or 8.9 kb (lanes 5, 6) insize were amplified by PCR. M: DNA sizing markers (HindIII digest of λDNA).

DETAILED DESCRIPTION OF THE INVENTION

[0035] Overview

[0036] The present invention is directed to compositions and methods foruse in reverse transcriptase-polymerase chain reaction (RT-PCR)production and analysis of nucleic acids. In particular, the inventionprovides compositions comprising a variety of components in variouscombinations. Such components include one or more sulfur-containingmolecules or one or more acetate-containing molecules (or combinationsof one or more sulfur-containing molecules and one or moreacetate-containing molecules), one or more enzymes having reversetranscriptase activity, one or more DNA polymerases, one or moreprimers, one or more nucleotides and a suitable buffer. Thesecompositions may be used in the methods of the invention to produce,analyze, quantitate and otherwise manipulate nucleic acid moleculesusing a one- or two-step RT-PCR procedure.

[0037] Compositions

[0038] The buffer in the compositions of the invention provideappropriate pH and ionic conditions for the enzymes having reversetranscriptase activity and DNA polymerase enzymes. The nucleotides usedin the compositions (e.g., deoxyribonucleoside triphosphates (dNTPs)),and the primer nucleic acid molecules provide the substrates forsynthesis or amplification of nucleic acid molecules in accordance withthe invention. The compositions of the invention may also includeinhibition-relieving reagents to assist in overcoming inhibition inRT-PCR reactions.

[0039] Buffer and Ionic Conditions

[0040] The buffer and ionic conditions of the present compositions havebeen optimized to relieve RT-mediated inhibition of RT-PCR. Preferredcompositions of the invention comprise one or more sulfur-containingmolecules, which provide sulfur in ionic form such as sulfate ions,sulfite ions, sulfonate ions (e.g., p-toluenesulfonic acid) and thelike. Additional preferred compositions of the invention comprise one ormore acetate-containing molecules, or combinations of one or moresulfur-containing molecules and one or more acetate-containingmolecules.

[0041] The sulfur-containing molecules should be formulated into thecompositions to preferably provide a concentration of sulfur in thesolution of at least 18 mM, more preferably a concentration of at least19 mM and most preferably a concentration of at least 20 mM.Particularly preferred concentration ranges for sulfur in the presentcompositions include about 18 mM to about 500 mM, about 18 mM to about150 mM, about 18 mM to about 100 mM about 18 mM to about 75 mM, about 18mM to about 50 mM or about 18 mM to about 40 mM, and most preferablyabout 18 mM to about 50 mM.

[0042] The sulfur-containing molecules are preferably formulated intothe present compositions in the form of one or more salts or buffers.Examples of suitable sulfur-containing salts according to the inventioninclude, but are not limited to, ammonium sulfate, magnesium sulfate,manganese sulfate, potassium sulfate, sodium sulfate and the like. Mostpreferred are ammonium sulfate, magnesium sulfate and manganese sulfate.Examples of suitable sulfur-containing buffers according to theinvention include, but are not limited to, TRIS-sulfate and othersulfuric acid-based buffers, as well as sulfonic acid-based buffers suchas AMPSO(3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid),BES (N,N-bis[2-hydroxyethyl]-2-aminomethanesulfonic acid), MOPS(3-N-morpholino)-propanesulfonic acid), MOPSO(3-N-morpholino)-2-hydroxypropanesulfonic acid, TES(2-{([tris-(hydroxymethyl)-methyl]amino}ethanesulfonic acid), HEPES(N-2-hydroxyethylpiperazine-N′-2-ethansulfonic acid), HEPPS(N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid), HEPPSO(N-2-hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid), TAPS (TES(3-{([tris-(hydroxymethyl)methyl]amino}propanesulfonic acid, CHES(2-(N-cyclo-hexylamino)ethanesulfonic acid), MES(2-N-morpholino)ethanesulfonic acid, PIPES(piperazine-N,N′-bis-2-ethanesulfonic acid), POPSO(piperazine-N,N′-bis[2-hydroxy]propanesulfonic acid), TAPS(N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid), TAPSO(3-[N-tris{hydroxymethyl}methylamino]-2-hydroxypropanesulfonic acid),ACES (N-2-acetamide-2-aminoethane sulfonic acid), DIPSO(3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), CAPSO(3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid) and CAPS(3-[cyclohexylamino]propanesulfonic acid). Other sulfur-containing ionicsalts and buffers, and other sulfur-containing molecules, suitable foruse in the compositions of the invention will be apparent to one ofordinary skill in the art.

[0043] The acetate-containing molecules should be formulated into thecompositions to preferably provide a concentration of acetate ion in thesolution of about 1 mM to about 500 mM. Particularly preferredconcentration ranges for acetate ion in the present compositions includeabout 1 mM to about 500 mM, about 5 mM to about 250 mM, about 10 mM toabout 200 mM, about 25 mM to about 150 mM, about 50 mM to about 100 mMor about 60 mM.

[0044] The acetate-containing molecules are preferably formulated intothe present compositions in the form of one or more salts or buffers.Examples of suitable acetate-containing salts according to the inventioninclude, but are not limited to, ammonium acetate, magnesium acetate,manganese acetate, potassium acetate, sodium acetate and the like.Examples of suitable acetate-containing buffers according to theinvention include, but are not limited to, TRIS-acetate(tris[{hydroxymethyl}]aminomethane] acetate), ADA(N-[2-acetamido]-2-iminodiacetic acid), and imidazole acetate(2-hydroxy-3-[4-imidazolyl]-propionic acid).

[0045] In accordance with the invention one or more potassium-containingmolecules may be formulated into the present compositions to substitutefor, and thereby reduce the concentration requirement for, sulfur. Inthis aspect of the invention, the addition of both sulfur-containingmolecules and potassium-containing molecules decreases the concentrationrequirement for sulfur by about 13-75%, preferably by about 25-50%. Forexample, when potassium-containing molecules are formulated into thepresent compositions, the concentration of sulfur-containing moleculesmay be reduced from about 18 mM to about 2-14 mM, or preferably to about4-9 mM. It will be understood, of course, that the one or more potassiumions may also be used in the above-described compositions of theinvention that contain one or more acetate-containing molecules insteadof, or in addition to, the one or more sulfur-containing molecules.

[0046] The potassium-containing molecules should be formulated into thecompositions at a preferred concentration of at least 2 mM, preferablyat least 5 mM, still more preferably at least 10 mM, and most preferablyat least 20 mM. Particularly preferred concentration ranges ofpotassium-containing molecules in the present compositions include about2 mM to about 500 mM, about 2 mM to about 200 mM, about 2 mM to about100 mM, about 2 mM to about 75 mM, about 2 mM to about 50 mM, about 2 mMto about 40 mM, about 2 mM to about 30 mM, about 2 mM to about 20 mM andabout 2 mM to about 10 mM.

[0047] The potassium-containing molecules are preferably formulated intothe present compositions in the form of one or more salts or buffers.Examples of suitable potassium salts according to the invention include,but are not limited to, potassium sulfate, potassium sulfite, potassiumchloride, potassium nitrate and the like. Most preferred are potassiumchloride, potassium sulfate and potassium acetate. Preferred potassiumbuffers according to the invention include, but are not limited to,potassium phosphate (monobasic), potassium phosphate (dibasic) and thelike. Other potassium salts and buffers, and other potassium-containingmolecules, suitable for use in the present compositions will be apparentto one of ordinary skill in the art.

[0048] Molecules and buffers containing sulfur, acetate or potassiumthat are suitable for use in the present compositions are availablecommercially from a wide variety of sources, e.g., from Sigma (St.Louis, Mo.).

[0049] Reverse Transcriptase Enzymes

[0050] The compositions of the present invention also comprise enzymeshaving reverse transcriptase activity. According to the presentinvention, the enzymes having reverse transcriptase activity are MoloneyMurine Leukemia Virus (M-MLV) reverse transcriptases. Preferred enzymesfor use in the invention include those that are substantially reduced inRNase H activity. By an enzyme “substantially reduced in RNase Hactivity” is meant that the enzyme has less than about 20%, morepreferably less than about 15%, 10% or 5%, and most preferably less thanabout 2%, of the RNase H activity of a wildtype or RNase H⁺ enzyme suchas wildtype M-MLV reverse transcriptase. The RNase H activity may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.

[0051] Particularly preferred enzymes for use in the invention include,but are not limited to, M-MLV reverse transcriptase (RNase H⁻ orsubstantially reduced in RNase H activity), and RSV reversetranscriptase (RNase H⁻ or substantially reduced in RNase H activity).Enzymes having reverse transcriptase activity are commercially available(for example, SUPERSCRIPT™, SUPERSCRIPT II™ and M-MLV, available fromLife Technologies, Inc.; Rockville, Md.).

[0052] DNA Polymerases

[0053] The compositions of the invention also comprise one or more DNApolymerases, which are preferably thermostable DNA polymerases. TheseDNA polymerases may be isolated from natural or recombinant sources, bytechniques that are well-known in the art (See WO 92/06200, U.S. Pat.Nos. 5,455,170 and 5,466,591, WO 96/10640 and U.S. patent applicationSer. No. 08/370,190, filed Jan. 9, 1995, the disclosures of all of whichare incorporated herein by reference), from a variety of thermophilicbacteria that are available commercially (for example, from AmericanType Culture Collection, Rockville, Md.) or may be obtained byrecombinant DNA techniques (see, e.g., WO 96/10640 and U.S. patentapplication Ser. No. 08/370,190, filed Jan. 9, 1995). Suitable for useas sources of thermostable polymerases or the genes thereof forexpression in recombinant systems are the thermophilic bacteria Thermusthermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcuswoosii and other species of the Pyrococcus genus, Bacillussterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum,Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoganeapolitana, Thermotoga maritima and other species of the Thermotogagenus, and Methanobacterium thermoautotrophicum, and mutants, variantsor derivatives thereof. It is to be understood, however, thatthermostable DNA polymerases from other organisms may also be used inthe present invention without departing from the scope or preferredembodiments thereof. As an alternative to isolation, thermostable DNApolymerases are available commercially from, for example, LifeTechnologies, Inc. (Rockville, Md.), New England BioLabs (Beverly,Mass.), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, Calif.),Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and Perkin ElmerCetus (Norwalk, Conn.).

[0054] Particularly preferred thermostable DNA polymerases for use inthe compositions and methods of the present invention include, but arenot limited to, Taq, Tne, Tma, Tli/VENT™, DEEPVENT™, Pfu, Pwo, Tfi orTth DNA polymerases, or mutants or derivatives thereof Taq DNApolymerase is commercially available, for example from LifeTechnologies, Inc. (Rockville, Md.), or may be isolated from its naturalsource, the thermophilic bacterium Thermus aquaticus, as describedpreviously (U.S. Pat. Nos. 4,889,818 and 4,965,188). Tne DNA polymerasemay be isolated from its natural source, the thermophilic bacteriumThermotoga neapolitana (See WO 96/10640 and U.S. patent application Ser.No. 08/370,190, filed Jan. 9, 1995), and Tma DNA polymerase from itsnatural source, the thermophilic bacterium Thermotoga maritima (See U.S.Pat. No. 5,374,553, the disclosure of which is incorporated herein byreference). Methods for producing mutants and derivatives ofthermophilic DNA polymerases, particularly of Tne and Tma polymerases,are disclosed in co-pending U.S. patent application Ser. No. 08/689,807of Deb K. Chatterjee, and in co-pending U.S. patent application Ser. No.08/689,818 of Deb K. Chatterjee and A. John Hughes, both filed Sep. 6,1996, which are incorporated by reference herein in their entirety. Tfi,Tli/VENT™ and DEEPVENT™ are available commercially (e.g., from NewEngland BioLabs; Beverly, Mass.), or may be produced as described (Bej,A. K., and Mahbubani, M. H., in: PCR Technology: Current Innovations,Griffin, H. G., and Griffin, A. M., eds., CRC Press, pp. 219-237 (1994)for Tli/VENT™; Flaman, J. -M., et al., Nucl. Acids Res. 22(15):3259-3260(1994) for DEEPVENT™). Thermostable DNA polymerases are preferably addedto the present compositions at a final concentration in solution ofabout 0.1-200 units per milliliter, about 0.1-50 units per milliliter,about 0.1-40 units per milliliter, about 0.1-36 units per milliliter,about 0.1-34 units per milliliter, about 0.1-32 units per milliliter,about 0.1-30 units per milliliter, or about 0.1-20 units per milliliter,and most preferably at a concentration of about 20 units per milliliter.

[0055] In preferred compositions of the invention, the concentration ofDNA polymerases is determined as a ratio of the concentration of theenzymes having reverse transcriptase activity. Thus, in particularlypreferred compositions the unit ratio of the reverse transcriptaseenzymes to the DNA polymerase enzymes ranges from about 0.2:2 to about500:2, preferably from about 0.5:2 to about 250:2 and most preferably aratio of greater than 3:2. Of course, other suitable ratios of unitactivities of reverse transcriptase enzymes to DNA polymerases suitablefor use in the invention will be apparent to one of ordinary skill inthe art.

[0056] Inhibition-Relieving Reagents

[0057] In accordance with the methods of the invention, one or moreadditional inhibition-relieving agents may optionally be added to thepresent compositions to assist in overcoming the inhibition of RT-PCRreactions by RTs such as M-MLVRT. Preferred inhibition-relieving agentsfor use in the present compositions include peptides, polypeptides andproteins such as (but not limited to) human serum albumin, bovine serumalbumin, ovalbumin, Albumax, casein, gelatin, collagen, globulin,lysozyme, transferrin, myoglobin, hemoglobin, α-lactalbumin, fumarase,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), amyloglucosidase,carbonic anhydrase, β-lactoglobulin, aprotinin, soybean trypsininhibitor, trypsinogen, phosphorylase b, myosin, actin, β-galactosidase,catalase, tryptic soy digests, tryptose, lectins and the like, orfragments or derivatives thereof. Particularly preferred for use in thecompositions and methods of the invention are bovine serum albumin,human serum albumin, Albumax and casein. Peptides, polypeptides orproteins are preferably added to the compositions to give a finalconcentration in the solution of about 0.01 to about 100 μg/ml,preferably about 0.1 to about 100 μg/ml, more preferably about 1 toabout 50 μg/ml and most preferably about 2 to about 20 μg/ml.

[0058] dNTPs

[0059] The compositions of the invention further comprise one or morenucleotides (e.g., deoxynucleoside triphosphates (dNTPs)). Thenucleotide components of the present compositions serve as the “buildingblocks” for newly synthesized nucleic acids, being incorporated thereinby the action of the reverse transcriptases or DNA polymerases. Examplesof nucleotides suitable for use in the present compositions include, butare not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP,α-thio-dATP, α-thio-dTTP, α-thio-dGTP, α-thio-dCTP or derivativesthereof, all of which are available commercially from sources includingLife Technologies, Inc. (Rockville, Md.), New England BioLabs (Beverly,Mass.) and Sigma Chemical Company (Saint Louis, Mo.). The dNTPs may beunlabeled, or they may be detectably labeled by coupling them by methodsknown in the art with radioisotopes (e.g., ³H, ¹⁴C, ³²P or ³⁵S),vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein,rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels,dioxigenin and the like. Labeled dNTPs may also be obtainedcommercially, for example from Life Technologies, Inc. (Rockville, Md.)or Sigma Chemical Company (Saint Louis, Mo.). In the presentcompositions, the dNTPs are added to give a working concentration ofeach dNTP of about 10-1000 micromolar, about 10-500 micromolar, about10-250 micromolar, or about 10-100 micromolar, and most preferably aconcentration of about 100 micromolar.

[0060] Primers

[0061] In addition to nucleotides, the present compositions comprise oneor more primers which facilitate the synthesis of a first DNA moleculecomplementary to all or a portion of an RNA template (e.g., asingle-stranded cDNA molecule). Such primers may also be used tosynthesize a DNA molecule complementary to all or a portion of the firstDNA molecule, thereby forming a double-stranded cDNA molecule.Additionally, these primers may be used in amplifying nucleic acidmolecules in accordance with the invention. Such primers include, butare not limited to, target-specific primers (which are preferablygene-specific primers), oligo(dT) primers, random primers or arbitraryprimers. Additional primers that may be used for amplification of theDNA molecules according to the methods of the invention will be apparentto one of ordinary skill in the art.

[0062] Methods of RT-PCR

[0063] In the RT-PCR reaction, the reaction mixtures are incubated at atemperature sufficient to synthesize a DNA molecule complementary to allor portion of the RNA template. Such conditions typically range fromabout 20° C. to 75° C., more preferably from about 35° C. to 60° C. andmost preferably from about 45° C. to about 55° C. After the reversetranscription reaction, the reaction is incubated at a temperaturesufficient to amplify the synthesized DNA molecule. Preferably theamplification is accomplished via one or more polymerase chain reactions(PCRs). Preferred conditions for amplification comprise thermocycling,which may comprise alternating heating and cooling of the mixturesufficient to amplify the DNA molecule and which most preferablycomprises alternating from a first temperature range of from about 90°C. to about 100° C., to a second temperature range of from about 45° C.to about 75° C., more preferably from about 50° C. to about 75° C. orfrom about 55° C. to about 75° C., and most preferably from about 65° C.to about 75° C. According to the invention, the thermocycling may beperformed any number of times, preferably from about 5 to about 80times, more preferably greater than about 10 times and most preferablygreater than about 20 times.

[0064] The compositions and methods of the present invention may also beused for the production, analysis and quantitation of large nucleic acidmolecules (e.g., by “long PCR” or “long RT-PCR”), preferably nucleicacid molecules that are larger than about 4-8 kilobases in size, morepreferably larger than about 5-7 kilobases in size, and most preferablynucleic acid molecules that are larger than about 7 kilobases in size.In this aspect of the invention, combinations of DNA polymerases,preferably mixtures of one or more DNA polymerases lacking 3′-5′exonuclease activity (i.e., a “3′ exo⁻” polymerase) with one or more DNApolymerases having 3′-5′ exonuclease activity (i.e., a “3′ exo⁺”polymerase), may be added to the compositions of the invention (see U.S.Pat. No. 5,436,149; see also co-pending U.S. patent application Ser. No.08/801,720, of Ayoub Rashtchian and Joseph Solus, filed Feb. 14, 1997,and the co-pending U.S. Patent Application of Ayoub Rashtchian andJoseph Solus entitled “Stable Compositions for Nucleic AcidAmplification and Sequencing,” filed Mar. 27, 1998, the disclosures ofall of which are incorporated herein in their entireties). Preferred 3′exo⁻ and 3′ exo⁺ polymerases for use in this aspect of the invention arethermostable 3′ exo⁻ and 3′ exo⁺ polymerases. Particularly preferred 3′exo⁻ polymerases include, but are not limited to, Taq, Tne(exo⁻),Tma(exo⁻), VENT(exo⁻)™, DEEPVENT(exo⁻)™, Pfu (exo⁻) and Pwo(exo⁻)polymerases, or mutants, variants or derivatives thereof, which arepreferably added to the compositions of the invention at a concentrationin the solution of about 0.1-200 units per milliliter, about 0.1-50units per milliliter, about 0.1-40 units per milliliter, about 0.1-36units per milliliter, about 0.1-34 units per milliliter, about 0.1-32units per milliliter, about 0.1-30 units per milliliter, or about 0.1-20units per milliliter, and most preferably at a concentration of about 20units per milliliter. Particularly preferred 3′ exo⁺ polymerasesinclude, but are not limited to, VENT™, Pfu, Pwo, Tne, Kod and Tma, andmost preferably DEEPVENT™, DNA polymerases, which should be added to themixtures in sufficient quantity to give a final working concentration ofabout 0.0002-200 units per milliliter, about 0.002-100 units permilliliter, about 0.002-20 units per milliliter, about 0.002-2.0 unitsper milliliter, about 0.002-1.6 units per milliliter, about 0.002-0.8units per milliliter, about 0.002-0.4 units per milliliter, or about0.002-0.2 units per milliliter, most preferably at concentrations ofabout 0.40 units per milliliter. These thermostable DNA polymerases areavailable commercially, for example from Life Technologies, Inc.(Rockville, Md.), New England BioLabs (Beverly, Mass.), Finnzymes Oy(Espoo, Finland), Stratagene (La Jolla, Calif.), Boehringer MannheimBiochemicals (Indianapolis, Ind.) and Perkin-Elmer Cetus (Norwalk,Conn.). The mixtures of the compositions of the invention and the 3′exo⁻ and 3′ exo⁺ polymerases may be used in the methods of the inventionto result in enhanced sensitivity of detection, and yield, of largeRT-PCR products.

[0065] It will be readily apparent to one of ordinary skill in therelevant arts that other suitable modifications and adaptations to themethods and applications described herein are obvious and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLE 1 Inhibition of RT-PCR by RT

[0066] To examine inhibition of RT-PCR amplification by RT, CAT RNA wasused as a template. RT-PCR reactions were conducted in a 50 μl finalvolume in PCR buffer (20 mM Tris-HCl, 50 mM KCl) or ELONGASE buffer (60mM Tris-SO₄ (pH 9.1), 18 mM (NH₄)₂SO₄; total sulfur concentrationapproximately 23 mM) containing 20 units of M-MLV(H⁻) RT, 1 mMdithiothreitol (DTT), 0.2 mM each of sense and antisense primers, 0.2 mMdNTPs, 1.25 mM MgCl₂, 2 units of Taq and various amounts of CAT mRNAs(0, 10, 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ copies per reaction). RT-PCRconditions were one cycle of 45° C. for 30 minutes and 94° C. for 2minutes, followed by 40 cycles of 94° C. for 15 seconds/60° C. 30seconds and then 72° C. for 5 minutes.

[0067] Upon analysis of the amplification products by 1.5% agarose gelelectrophoresis (FIG. 1), significant amplification of the 500 bp targetsequence was observed in reactions carried out in ELONGASE buffer using106 or 107 copies of CAT mRNA template FIG. 1, lanes 8 and 9). Incontrast, no significant PCR product was observed for reactions carriedout in standard PCR buffer at all template concentrations (FIG. 1, lanes18, 19). These results indicate that the presence of RT in PCR reactionmixtures inhibits the amplification reaction, but that this inhibitionis at least partially relieved using ELONGASE buffer.

EXAMPLE 2 Role of Sulfur in Relief of RT Inhibition in PCR Amplification

[0068] To determine if the sulfate ion in ELONGASE buffer might be a keycomponent for the relief of RT inhibition observed in Example 1, variousreaction parameters such as pH, ionic conditions and buffer compositionwere studied in detail. RT-PCR reactions were carried out in a 50 μlfinal volume containing 1 mM DTT, 0.2 mM dNTPs, 1.5 mM MgSO₄, 0.2 mMeach of sense and antisense human β-actin primers, 1 pg of total HeLaRNA template, 2 units of Taq DNA polymerase and 10 units M-MLV(H⁻) RT inbuffered salt solutions comprising various ionic and buffer conditions.RT-PCR conditions were 30 minutes at 45° C. and 2 minutes at 94° C.,followed by 40 cycles of 94° C. for 15 seconds/55° C. for 30 seconds/68°C. for 90 seconds, and one final extension for 5 minutes at 72° C. Theability to detect an amplified 1,026-bp RT-PCR product from 1 pg oftotal HeLa RNA was used as an assessment of the amplification successunder the specific reaction conditions.

[0069] Upon analysis of 1,026-bp b-actin RT-PCR products in 1% agarosegel electrophoresis, the results shown in Tables 1-7 were obtained.

[0070] Table 1: Compositions comprising Tris-HCl (pH 8.5-9.3)demonstrated a sensitivity of about 1 pg total HeLa RNA when 18 mM(NH₄)₂SO₄ was present. However, this increased sensitivity was obtainedover a broader pH range (pH 7.8-9.3) when Tris-SO₄, buffer was used inplace of Tris-HCl. TABLE 1 Optimal pH in Tris-HCl and Tris-SO₄ Buffers.pH 7.5 7.8 8.0 8.3 8.5 8.8 9.0 9.3 9.5 Tris-HCl, 60mM¹ + + + + + + + + + (NH₄)₂SO₄, 18 mM + + + + + + + + + Sensitivity(Yield of − − − − + − ++ + − PCR Product) Tris-SO₄, 60mM + + + + + + + + + (NH₄)₂SO₄, 18 mM + + + + + + + + + Total Sulfur 4138 35 32 30 26 23 22 21 Concentration (mM)² Sensitivity (Yield of− + + + + + ++ + − PCR Product)

[0071] Table 2: In order to detect the 1,026-bp RT-PCR product incompositions comprising Tris-HCl buffer, the inclusion of 20 mM(NH₄)₂SO₄ in the compositions was essential. However, if Tris-SO₄ buffer(20-80 mM, pH 8.0-9.0) was used in place of Tris-HCl, the inclusion of(NH₄)₂SO₄ was not required. TABLE 2 Requirement for Sulfur andPotassium. pH 9.0 8.5 8.0 Tris-HCl,60 + + + + + + + + + + + + + + + + + + mM (NH₄)₂SO₄, 0 10 20 40 60 80 010 20 40 60 80 0 10 20 40 60 80 mM Sensitivity − − + − − − − − − − − − −− − − − − (Yield of PCR Product) pH 9.0 8.5 8.0 Tris-SO₄, mM 20 40 60 80100 20 40 60 80 100 20 40 60 80 100 KCl, 40mM + + + + + + + + + + + + + + + Total Sulfur 20 21 23 25  26 22 26 3034  38 24 29 35 41  46 Concentration (mM)² Sensitivity − − + + − + + + +− − − − − (Yield of PCR Product)

[0072] Table 3: Requirement for sulfur for sensitive detection of 1,026bp RT-PCR product was also demonstrated by use of taurine (NH₂CH₂SO₃H),which contains sulfur ion. Taurine relieved the RT-mediated inhibitionof RT-PCR about as well as did ammonium sulfate. TABLE 3 Requirement forSulfur. Tris-HCl, 60 mM (pH + + + + + + + + + + 9.0) [Taurine], mM 0 1020 40 60 0 0 0 0 0 [Ammonium Sulfate], 0 0 0 0 0 0 10 20 40 60 mMSensitivity (yield of − + ++ ++ ++ − + ++ − − PCR product) + + + +

[0073] Table 4: The increased detection sensitivity of the Tris-SO₄buffer system shown in Table 2 (60 mM, pH 8.5-9.0) could be furtherenhanced by the addition of 20-40 mM KCl, indicating thatpotassium-containing molecules not only were suitable substitutes forsulfur-containing molecules in the present compositions, but may alsoenhance the sensitivity of the RT-PCR reaction in their own right.

[0074] Table 5: Similar detection sensitivity and further enhancement bythe addition of KCl was obtained in the Tris-taurine buffer system.TABLE 4 Role of Potassium in Suboptimal Concentrations of Sulfur. pH 9.08.5 8.0 Tris-SO₄, + + + + + + + + + + + + + + + + + + 60 mM (NH₄)₂SO₄, 010 20 40 60  80 0 10 20 40 60  80 0 10 20 40 60  80 mM Sensitivity − −++ − − − − + + − − − − − − − − − (Yield of PCR Product)Tris-SO₄, + + + + + + + + + + + + + + + + + + 60 mM KCl, mM 0 20 40 6080 100 0 20 40 60 80 100 0 20 40 60 80 100 Sensitivity − − + + + − − −+++ +++ − − − − − − − − (Yield of PCR Product)

[0075] TABLE 5 Requirement for Sulfur and Potassium. [Tris- 60 60 60 6060 60 60 60 60 60 100 200 60 60 60 60 60 taurine], mM (pH 8.9) [Ammonium0 10 20 40 60 0 0 0 0 0 0 0 0 0 0 0 0 sulfate], mM [Taurine], 0 0 0 0 00 0 0 0 0 0 0 0 10 20 40 60 mM [KCl], mM 0 0 0 0 0 0 10 20 40 60 0 0 0 00 0 0 Sensitivity − +++ +++ − − − + ++ +++ +++ + ++ − + ++ +++ +++(yield of PCR product)

[0076] Table 6: The addition of NH₄Cl in place of (NH₄)₂SO₄ in thepresent compositions did not relieve the RT-mediated inhibition ofRT-PCR, indicating that sulfur-containing molecules are key componentsfor the relief of RT inhibition in RT-PCR. TABLE 6 Requirement forSulfur. Detection Sensitivity (Yield of PCR Product) Buffer Composition(Additive to (NH₄)₂SO₄, Tris-HCl, 60 mM) 20 mM NH₄Cl, 40 mM MagnesiumSulfate, 1.5 mM + − Magnesium Acetate, 1.5 mM + − Magnesium Chloride,1.5 mM + −

[0077] Table 7: Requirement for sulfur ion for relief of RT-mediatedinhibition of PCR was less stringent in Tris-acetate buffer systems thanin Tris-sulfate buffer systems. In the Tris-acetate buffer system RT-PCRproducts of 1,026 bp size were able to be observed even in the absenceof sulfur ions. TABLE 7 Requirement for Sulfur and Effect of Buffers.Tris-SO₄, 60 mM + + + + + − − − − − (pH 9.0) Tris-acetate, − − − −− + + + + + 60 mM (pH 8.4) [(NH₄)₂SO₄], mM 0 10 20 40 80 0 10 20 40 80Sensitivity (yield − ++ +++ − − + ++ +++ − − of PCR product)

EXAMPLE 3 Role of Bovine Serum Albumin (BSA) in RT-PCR

[0078] To investigate other reaction components which might relieve theRT-mediated inhibition of RT-PCR, BSA was added to the presentcompositions. Compositions were formulated comprising increasing amountsof M-MLV(H⁻) RT (from 10 units to 260 units) and various amount of BSA,and these compositions used in RT-PCR reactions. Reactions wereconducted in a 50 μl final volume containing 60 mM Tris-SO₄ (pH 9.1), 18mM (NH₄)₂SO₄, 0.2 mM dNTPs, 1.2 mM MgSO₄, 0.02 mM DTT, 0.2 mM each ofhuman β-actin CAT mRNA sense and antisense primers, 2 units of Taq DNApolymerase and 100 pg of total HeLa RNA template for β-actinamplification, or 10⁵ copies of total HeLa RNA template for CATamplification. RT-PCR conditions were 30 minutes at 45° C. and twominutes at 94° C., followed by 40 cycles of 94° C. for 15 seconds/55° C.for 30 seconds/68° C. for 90 seconds, and then one final extension offive minutes at 72° C.

[0079] Upon analysis of the 1,026-bp β-actin RT-PCR products in 1%agarose gel electrophoresis (FIG. 2A), RT inhibition was not observedwith 10 units of RT, but increasing amounts of RT (135 units-260 units)inhibited RT-PCR completely. However, such RT inhibition was relieved bythe addition of 200-1000 ng of BSA per reaction (i.e., a final BSAconcentration of about 4-20 μg/ml). Similar results were obtained fromthe analysis of the 653-bp CAT RT-PCR products (FIG. 2B), where only aslightly higher amount of BSA (300-1000 ng per reaction i.e., a finalBSA concentration of about 6-20 μg/ml) was required for the relief of RTinhibition. Together, these results indicate that the incorporation ofproteins such as BSA into the compositions of the invention may assistin overcoming the inhibition of RT-PCR caused by RT.

EXAMPLE 4 Performance of M-MLV, AMV, M-MLV(H⁻) for RT-PCR in Sulfur- andBSA-containing Buffer

[0080] To study the performance of various RTs in RT-PCR, AMV-RT,M-MLV-RT, and M-MLV(H⁻) RT were used in the present compositions. RT-PCRreactions for M-MLV-RT and M-MLV H⁻RT (Superscript II) were conducted ina 50 μl final volume containing 60 mM Tris-SO₄(pH 9.1), 18 mM (NH₄)₂SO₄,0.2 mM dNTPs, 250 ng BSA, 1.0 mM DTT, 0.2 mM each of CAT sense andantisense primers, 2 units of Taq DNA polymerase, 1.5 mM MgSO₄, andvarious amount of CAT RNA template (0, 10³, 10⁴ or 10⁵ copies perreaction). The compositions containing AMV-RT were the same except thatthey lacked BSA. For each reaction, 5 units each of AMV-RT, M-MLV-RT orM-MLV(H⁻) RT were used. RT-PCR cycling conditions were 30 minutes at 45°C. and 2 minutes at 94° C., followed by 40 cycles of 94° C. for 15seconds/60° C. for 30 seconds, and then a final extension of fiveminutes at 72° C.

[0081] As shown in FIG. 3, upon analysis of 500-bp CAT RT-PCR productsin 1.5% agarose gel electrophoresis the reactions performed with AMV-RT,M-MLV-RT, and M-MLV(H⁻)-RT demonstrated efficient and sensitive RT-PCRproduct yield, with no inhibition of the RT-PCR reactions observed underany of the reaction conditions.

EXAMPLE 5 RT-PCR Amplification of Long Nucleic Acid Templates

[0082] Having demonstrated the simplicity and sensitivity of the presentmethods in RT-PCR amplification of templates up to 3.5 kb in size, theefficacy of the invention in amplifying mRNAs up to 8.9 kb was examined.

[0083] Total HeLa RNA was isolated with TRIzol® Reagent (LifeTechnologies, Inc.; Rockville, Md.) and amplified as above. To examinepossible temperature effects, identical reactions were assembled andincubated at 45° C. to 55° C. in duplicate. The total HeLa RNA usedvaried from 1 ng to 100 ng depending on the abundance of the mRNA. Afterthe RT incubation for 30 minutes, the reactions were incubated at 94° C.for two minutes, followed by 40 cycles of 94° C. for 15 seconds, 55° C.for 30 seconds and 68° C. for 60 seconds, followed by a final 68° C.extension for five minutes.

[0084] For larger RT-PCR products, the magnesium concentration wasincreased to 1.8 mM from the standard 1.5 mM, and 1 μl of ELONGASEEnzyme Mix (Life Technologies, Inc.; Rockville, Md.) was added to eachreaction. The final 50 μl reaction consisted of 1× ELONGASE buffer with1.8 mM MgSO₄ and other salts, 200 μM of each dNTP, 2 μg/ml BSA, 0.2 μMof primers (GIBCO BRL Custom Primers; Life Technologies, Inc.,Rockville, Md.), 100 ng of total HeLa RNA, 1 μl of the RT/Taq enzyme mixof the invention and 1 μl of ELONGASE Enzyme Mix. For the experimentsusing the same template of primers, a master mix of buffer, enzymemixes, and primers or template was made to ensure consistency. Thesamples were incubated at 50° C. for 30 minutes and then 94° C. for twominutes. Amplification was performed with 40 cycles of 94° C. for 15seconds, 58° C. for 30 seconds and 68° C. for six to nine minutes (oneminute/kb). The RT-PCR products were resolved and visualized on 0.8 or1.0% (w/v) agarose-TAE gels containing ethidium bromide.

[0085] Temperature of the RT reaction. SUPERSCRIPT II RT has improvedtemperature stability compared to M-MLV-RT (Gerard, G., et al., FOCUS14:91 (1992)). To test the effect of temperature on RT-PCR products, 36primer sets (representing different genes from human, rat, plant, andone in vitro transcript) were examined. As shown in FIG. 4, substantialproduct yield and specificity were observed at 45° C. for many of thefragments examined; increased incubation temperature did not result inincreased yield or specificity. This result, however, was dependent uponthe specific template and primer chosen (FIG. 5): for sometemplate-primer combinations, the smearing of bands caused by misprimingthat was characteristic of RT reactions conducted at 45° C. disappearedwith increased temperature (FIG. 5; lanes 1-6), while in some cases asignificant increase in product yield was observed when the RT reactionswere conducted at elevated temperatures (FIG. 5; lanes 7-12).

[0086] Long RT-PCR Products. For studies of full-length coding sequencesor for amplification of long segments of RNA, the use of thecompositions of the present invention supplemented with ELONGASE EnzymeMix was tested. As shown in FIG. 6, the present system and Supplier A'skit were able to amplify a 2.8-kb product from 100 ng, but not from 10ng, of total RNA. The addition of ELONGASE Enzyme Mix to the presentsystem not only increased the product yield with 100 ng of total RNA,but also increased the sensitivity to allow amplification of longtemplates from 10 ng total RNA (FIG. 6; lanes 7-12). In contrast,Supplier A's kit was not able to amplify the 2.8-kb target from 10 ng oftotal RNA (FIG. 6; lanes 13-18) even though it contained a polymeraseenzyme mix designed for long templates.

[0087] The compositions and methods of the invention were also found tobe useful in amplifying large (>3-5 kb) RT-PCR products. As shown inFIG. 7, the compositions of the present invention, when supplementedwith ELONGASE Enzyme Mix, produced RT-PCR products up to 8.9 kb in size.The ladder of bands observed upon amplification with these compositionsis not uncommon for long RT-PCR, and the variation in product yields maybe partially due to the use of different primer sets. These resultsdemonstrate that the addition of ELONGASE Enzyme Mix to the presentcompositions makes it possible to amplify long and rare mRNAs directlyfrom total RNA preparations by the methods of the invention. Further,the thermostability of SUPERSCRIPT II RT, facilitating RT-reactions attemperatures of up to 55° C., can increase the specificity and productyield for some templates and primer sets.

[0088] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0089] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

What is claimed is:
 1. A method for amplifying a nucleic acid molecule,said method comprising (a) mixing an RNA template with a compositioncomprising a Moloney murine leukemia virus (M-MLV) reversetranscriptase, one or more DNA polymerases and one or moresulfur-containing molecules at a concentration of at least 18 mM sulfur,to form a mixture; and (b) incubating said mixture under conditionssufficient to amplify a DNA molecule complementary to all or a portionof said RNA template.
 2. The method of claim 1, wherein said compositionfurther comprises one or more acetate-containing molecules.
 3. A methodfor amplifying a nucleic acid molecule, said method comprising (a)mixing an RNA template with a composition comprising a Moloney murineleukemia virus (M-MLV) reverse transcriptase, one or more DNApolymerases and one or more acetate-containing molecules at aconcentration of about 1 mM to about 500 mM, to form a mixture; and (b)incubating said mixture under conditions sufficient to amplify a DNAmolecule complementary to all or a portion of said RNA template.
 4. Themethod of claim 1 or claim 3, wherein said DNA polymerase is Taq DNApolymerase.
 5. The method of claim 1 or claim 3, wherein said DNApolymerases comprise a first DNA polymerase having 3′ exonucleaseactivity and a second DNA polymerase having substantially reduced 3′exonuclease activity.
 6. The method of claim 1 or claim 3, wherein theunit ratio of said reverse transcriptase to said DNA polymerases is fromabout 0.2:2 to about 500:2.
 7. The method of claim 6, wherein said ratiois from about 0.5:2 to about 250:2.
 8. The method of claim 6, whereinsaid ratio is greater than 3:2.
 9. The method of claim 1, wherein saidsulfur concentration is at least 19 mM.
 10. The method of claim 1,wherein said sulfur concentration is about 20 mM to about 50 mM.
 11. Themethod of claim 1, wherein the source of said sulfur is asulfur-containing buffer.
 12. The method of claim 1, wherein the sourceof said sulfur is a sulfur-containing salt.
 13. The method of claim 11,wherein said sulfur-containing buffer is TRIS-sulfate(tris[{(hydroxymethyl}]aminomethane] sulfate).
 14. The method of claim12, wherein said sulfur-containing salt is selected from the groupconsisting of ammonium sulfate, magnesium sulfate and manganese sulfate.15. The method of claim 2 or claim 3, wherein the concentration of saidacetate-containing molecule is about 60 mM.
 16. The method of claim 2 orclaim 3, wherein said acetate-containing molecule is anacetate-containing salt.
 17. The method of claim 2 or claim 3, whereinsaid acetate-containing molecule is an acetate-containing buffer. 18.The method of claim 16, wherein said acetate-containing salt is selectedfrom the group consisting of ammonium acetate, magnesium acetate,manganese acetate, sodium acetate and potassium acetate.
 19. The methodof claim 17, wherein said acetate-containing buffer is selected from thegroup consisting of TRIS-acetate (tris[{hydroxymethyl}]aminomethane]acetate), ADA (N-[2-acetamido]-2-iminodiacetic acid), and imidazoleacetate (2-hydroxy-3-[4-imidazolyl]-propionic acid).
 20. The method ofclaim 1 or claim 3, wherein said mixture further comprises one or morenucleotides.
 21. The method of claim 20, wherein said nucleotides aredeoxyribonucleoside triphosphates or derivatives thereof ordideoxyribonucleoside triphosphates or derivatives thereof.
 22. Themethod of claim 20, wherein said deoxyribonucleoside triphosphates areselected from the group consisting of dATP, dUTP, dTTP, dGTP, and dCTP.23. The method of claim 1 or claim 3, wherein said mixture furthercomprises one or more oligonucleotide primers.
 24. The method of claim23, wherein said primer is an oligo(dT) primer.
 25. The method of claim23, wherein said primer is a target-specific primer.
 26. The method ofclaim 23, wherein said primer is a gene-specific primer.
 27. The methodof claim 1 or claim 3, wherein said reverse transcriptase issubstantially reduced in RNase H activity.
 28. The method of claim 1 orclaim 3, wherein said DNA polymerases are thermostable DNA polymerases.29. The method of claim 28, wherein said thermostable DNA polymerasesare selected from the group consisting of Tne, Tma, Taq, Pfu, Tth, Tfi,VENT, DEEPVENT, Pwo, Tfl, and mutants, variants and derivatives thereof.30. The method of claim 5, wherein said DNA polymerase having 3′exonuclease activity is selected from the group consisting of Pfu, Pwo,DEEPVENT, VENT, Tne, Tma, Kod, and mutants, variants and derivativesthereof.
 31. The method of claim 5, wherein said DNA polymerase havingsubstantially reduced 3′ exonuclease activity is selected from the groupconsisting of Taq, Tfl, Tth, and mutants, variants and derivativesthereof.
 32. The method of claim 1 or claim 3, wherein said incubatingstep (b) comprises (a) incubating said mixture at a temperature and fora time sufficient to make a DNA molecule complementary to all or aportion of said RNA template; and (b) incubating said DNA moleculecomplementary to said RNA template at a temperature and for a timesufficient to amplify said DNA molecule.
 33. The method of claim 32,wherein said first step comprises incubating said mixture at atemperature from about 35° C. to about 60° C.
 34. The method of claim32, wherein said second step comprises thermocycling.
 35. The method ofclaim 34, wherein said thermocycling comprises alternating heating andcooling of the mixture sufficient to amplify said DNA molecule.
 36. Themethod of claim 35, wherein said thermocycling comprises alternatingfrom a first temperature range of from about 90° C. to about 100° C., toa second temperature range of from about 45° C. to about 75° C.
 37. Themethod of claim 36, wherein said second temperature range is from about65° C. to about 75° C.
 38. The method of claim 34, wherein saidthermocycling is performed greater than 10 times.
 39. The method ofclaim 34, wherein said thermocycling is performed greater than 20 times.40. The method of claim 1 or claim 3, wherein said reverse transcriptaseis SuperScript I or SuperScript II.
 41. The method of claim 1 or claim3, wherein said amplification is not substantially inhibited.
 42. Amethod for amplifying a nucleic acid molecule comprising: (a) mixing anRNA template with a composition comprising a Moloney murine leukemiavirus (M-MLV) reverse transcriptase, one or more DNA polymerases, one ormore sulfur-containing molecules and one or more potassium-containingmolecules, to form a mixture; and (b) incubating said mixture underconditions sufficient to amplify a DNA molecule complementary to all ora portion of said RNA template.
 43. The method of claim 42, wherein saidcomposition further comprises one or more acetate-containing molecules.44. A method for amplifying a nucleic acid molecule comprising: (a)mixing an RNA template with a composition comprising a Moloney murineleukemia virus (M-MLV) reverse transcriptase, one or more DNApolymerases, one or more acetate-containing molecules and one or morepotassium-containing molecules, to form a mixture; and (b) incubatingsaid mixture under conditions sufficient to amplify a DNA moleculecomplementary to all or a portion of said RNA template.
 45. The methodof claim 42 or claim 44, wherein said reverse transcriptase issubstantially reduced in RNase H activity.
 46. The method of claim 42 orclaim 44, wherein said DNA polymerases are selected from the groupconsisting of Tne, Tma, Taq, Pfu, Tth, Tfi, VENT, DEEPVENT, Pwo, Tfl,and mutants, variants and derivatives thereof.
 47. A method foramplifying a nucleic acid molecule comprising: (a) contacting an RNAtemplate with a mixture comprising a Moloney murine leukemic virus(M-MLV) reverse transcriptase and one or more DNA polymerases, whereinthe unit ratio of said reverse transcriptase to said DNA polymerases isgreater then 3:2, and (b) incubating said mixture under conditionssufficient to amplify a DNA molecule complementary to all or a portionof said RNA template.
 48. A composition comprising a Moloney murineLeukemic virus (M-MLV) reverse transcriptase, one or more DNApolymerases and at least 18 mM sulfur.
 49. The composition of claim 47,further comprising one or more acetate-containing molecules.
 50. Acomposition comprising a Moloney murine Leukemic virus (M-MLV) reversetranscriptase, one or more DNA polymerases, and one or moreacetate-containing molecules.
 51. The composition of claim 49 or claim50, wherein the concentration of said one or more acetate-containingmolecules is about 1 mM to about 500 mM.
 52. The composition of claim51, wherein the concentration of said one or more acetate-containingmolecules is about 60 mM.
 53. A composition comprising a Moloney murineLeukemic virus (M-MLV) reverse transcriptase, one or more DNApolymerases, one or more sulfur-containing molecules, and one or morepotassium-containing molecules.
 54. The composition of claim 53, furthercomprising one or more acetate-containing molecules.
 55. A compositioncomprising a Moloney murine Leukemic virus (M-MLV) reversetranscriptase, one or more DNA polymerases, one or moreacetate-containing molecules, and one or more potassium-containingmolecules.
 56. A composition comprising a Moloney murine leukemic virus(M-MLV) reverse transcriptase and one or more DNA polymerases, whereinthe unit ratio of said reverse transcriptase to said DNA polymerases isgreater than about 3:2.